Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing useful metabolites by fermentation of a bacterium of the family Enterobacteriaceae, wherein the LysR-type protein-regulated expression system of the bacterium has been modified in such a way that functionality of said expression system is mediated by a coinducer, and as a result, the expression levels of the genes regulated by said expression system are enhanced. More specifically, the expression system and the method can be useful for improving the production of metabolites from synthetic pathways of L-amino acids, such as the branched-chain L-amino acid.
Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765) and alteration of regulatory regions such as promoter, leader sequence, and/or attenuator or others known to the person skilled in the art (see, for example, US20060216796 A1 and WO9615246 A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042 A1, EP0685555 A1 or U.S. Pat. Nos. 4,346,170, 5,661,012, and 6,040,160). For example, the mutant bacterial acetohydroxy-acid synthetase I (also referred to as acetolactate synthase I, hereinafter AHAS I) which is resistant to feedback inhibition by L-valine has been utilized for improving branched-chain L-amino acid production in corresponding L-amino acid producing strains (Russian Patent No. 2355763).
The biosynthesis of branched-chain L-amino acids (BCAAs), such as L-valine, L-leucine, and L-isoleucine, occurs through a branched biosynthetic pathway. Acetolactate synthase (the enzyme classification (EC) number 2.2.1.6) catalyzes the reaction in the first step in the pathway, which is common to all three amino acid's biosynthetic pathways. The reaction includes condensation of activated acetaldehyde (2-(α-hydroxyethyl)thiamine diphosphate) derived from pyruvate with either pyruvate or 2-oxobutanoate to yield 2-acetolactate (AL) or 2-aceto-2-hydroxybutanoate (AHB), respectively. AL is a precursor of L-valine and L-leucine, and AHB is a precursor of L-isoleucine. In Escherichia coli (E. coli), for example, reactions between pyruvate molecules, as well as reactions between pyruvate and 2-oxobutanoate, are catalyzed by the three AHAS isozymes, AHAS I, AHAS II, and AHAS III, which are encoded by the ilvBN, ilvGM, and ilvIH genes, respectively. AHAS I and AHAS III are the targets for the end-product inhibition (also referred to as feedback inhibition) by L-valine. The feedback inhibition by the end-product plays a major role in the physiological control of these pathways in bacteria.
The products of the AHAS catalyzed reaction, AL or AHB, are the substrates for 2-acetohydroxy acid isomeroreductase IlvC (EC 1.1.1.86) which is encoded by the ilvC gene, a member of the ilvYC operon. The ilvYC operon of E. coli is a prototypical LysR protein-regulated system which is the most common type of positive regulatory system in bacteria, and can be found in prokaryotic bacterial families ranging from Enterobacteriaceae to Rhizobiaceae (Rhee K. Y. et al., Proc. Nat. Acad. Sci. USA, 1999, 96:14294-14299). The ilvY gene encodes the LysR-type regulatory protein IlvY, a transcriptional regulator, which binds in a highly cooperative fashion to two tandem operator regions in the divergent-overlapping ilvYC promoter region (FIG. 1). Upon binding to the first operator region, the IlvY regulator negatively auto-regulates transcription from the ilvY promoter thus attenuating its own synthesis. Apart from this function, IlvY plays a pivotal role in activation of transcription of the ilvC gene. Activation of ilvC transcription requires binding of the IlvY regulator to the second operator region and additional binding of a coinducer such as 2-acetolactate (AL) or 2-aceto-2-hydroxybutanoate (AHB) to a preformed IlvY/DNA complex. Upon binding a coinducer, a conformational change in the protein/DNA complex occurs that remodels the −35 region of the ilvC promoter and drastically increases RNA polymerase binding capacity (Rhee K. Y. et al., J. Biol. Chem., 1998, 273:11257-11266).
In the L-valine and L-leucine biosynthesis, 2-acetolactate (AL) is converted by the IlvC protein into 2,3-dihydroxy-3-methylbutanoate (also referred to as 2,3-dihydroxy-isovalerate, DHIV) (FIG. 2). In L-isoleucine biosynthesis, 2-aceto-2-hydroxybutanoate (AHB) is converted by IlvC into 2,3-dihydroxy-3-methylpentanoate (also referred to as 2,3-dihydroxy-3-methylvalerate, DHMV).
Recently, auto-inducible gene expression systems were recognized as being very attractive for enhancing expression of a desired gene over routine genetic approaches. For example, the literature provides an artificially designed positive feedback-based gene expression system that can function as a genetic signal amplifier heightening the sensitivity to protein inducer signals as well as increasing maximum expression levels without the need for the external cofactor acyl homoserine lactone (AHL, also abbreviated as HSL) (Nistala G. J. et al., J. Biol. Eng., 2010, 4:4). The designed system utilizes a constitutively active variant of the quorum-sensing (QS) regulator LuxR (lux operon) from Vibrio fischeri, which is auto-inducer (AHL)-independent due to the Ala221Val point mutation (Sayut D. J. et al., Biochem. Biophys. Res. Commun., 2007, 363:667-673; Poellinger K. A. et al., FEMS Microbiol Lett., 1995, 129:97-101). A similar gene expression system with slight variations has been applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli (Bansal K. et al., J. Biol. Eng., 2010, 4:6).
The auto-inducible positively feedback regulated activation system based on quorum-sensing machinery from V. fischery (lux bioluminescence genes) and an endogenous source of an auto-inducer (HSL) have been utilized for expressing recombinant proteins such as as antigens to prepare pharmaceutical compositions (WO2010136897 A2).
However, no data has been reported to date describing a LysR-type protein-regulated gene(s) expression system modified in such a way that the functionality of said expression system is mediated by a coinducer, and the use thereof for production of useful metabolites from the biosynthetic pathway of L-amino acid such as branched-chain L-amino acids and/or a branched off pathway thereof.